Process for producing a bran pickles flavoring solution

ABSTRACT

The present invention relates to a novel flavoring solution for making pickles having a flavor of foods pickled in rice bran paste (hereinafter referred to as a bran pickles flavoring solution), a clear bran pickles flavoring solution, processes for producing said solutions, a novel microorganism belonging to the genus Corynebacterium used for the production of said flavoring solutions, and a process for producing a solution containing a lactone wherein the lactone is selectively extracted from a culture of a lactone-producing microorganism.  
     The present invention also relates to a novel bacterium of the genus Corynebacterium which has the ability to produce γ-dodecalactone and/or γ-dodecelactone and is useful for the production of said bran pickles flavoring solutions.

TECHNICAL FIELD

[0001] The present invention relates to a flavoring solution for makingpickles having a flavor of foods pickled in rice bran paste (hereinafterreferred to as a bran pickles flavoring solution), a clear bran picklesflavoring solution, processes for producing said flavoring solutions, aprocess for producing lactones which are essential components of saidflavoring solutions, and a novel bacterium belonging to the genusCorynebacterium which is useful for producing a lactone.

BACKGROUND ART

[0002] It is common to make foods pickled in rice bran paste(hereinafter referred to as bran pickles) at home. However, in order tomake bran pickles having a good flavor, a mature rice bran paste must beprepared by carrying out pickling of foods such as vegetables andstirring of the bran paste every day for more than several months. Thus,it takes a lot of efforts to take care of rice bran paste.

[0003] In order to obtain foods having a flavor of bran pickles withoutusing bran paste, a method has been proposed which comprises picklingfoods such as vegetables in a pickling solution having a flavor of branpickles, and a study has been made on the aromatic components andseasoning components of a mature rice bran paste [Nippon NogeikagakuKaishi, 57, 1113 (1983)]. However, a flavoring substance essential to adesirable bran pickles flavoring solution has not been specified yet,and thus there has not been established an industrial process forproducing a bran pickles flavoring solution containing such a compoundat a high concentration.

[0004] As aromatic components of bran paste, γ-lactones such asγ-dodecalactone, γ-dodecelactone and γ-decalactone are considered to beimportant. As for the production of precursors of γ-lactones, it isknown that a lactic acid bacterium [Gastroenterology, 62, 430 (1972)], abacterium belonging to the genus Corynebacterium [Agricultural andBiological Chemistry, 45, 2025 (1981)], a bacterium belonging to thegenus Pseudomonas [Archives of Biochemistry and Biophysics, 99, 249(1962)], etc. are capable of producing γ-dodecalactone precursor(10-hydroxyoctadecanoic acid).

[0005] Also known is a method for converting ricinoleic acid intoγ-decalactone using yeast belonging to the genus Saccharomyces, etc. toimprove the quality of castor oil containing ricinoleic acid (JapanesePublished Unexamined Patent Application No. 66991/85, Japanese PublishedUnexamined Patent Application No. 100508/85). Further, it is known thatγ-lactones can be produced by fermentation using Sporobolomyces oderus,Fusarium poae, etc. separately.

[0006] A clear bran pickles flavoring solution is preferred from thestandpoint of commercial value, because the image of a flavoringsolution as a commercial product is improved and there is no need forremoving suspended matters such as rice bran from the obtained pickles.

[0007] Previous methods for producing a clear bran pickles flavoringsolution include a method which comprises fermenting a dispersedsolution of rice bran (hereinafter referred to as a bran suspension) andsubjecting the fermented bran suspension to solid-liquid separation by afilter press, a rotary filter or a centrifuge to obtain a separatedsolution as a bran pickles flavoring solution. According to suchmethods, a clear bran pickles flavoring solution can be obtained.However, fat-soluble aromatic components such as γ-lactones are removedtogether with the residue in these methods, and therefore, a desirablebran pickles flavoring solution can not be obtained.

[0008] Further, when a fermented bran suspension is extracted with anaqueous solution of ethanol and the obtained extract is mixed with theabove separated solution to obtain a bran pickles flavoring solution,the fatty acids extracted with an aqueous solution of ethanol areprecipitated and suspended in the mixture, making the resultingflavoring solution turbid. Thus, a clear bran pickles flavoring solutioncan not be obtained.

[0009] There have been reported a process for producing a bran picklingcomposition by fermentation using a lactic acid bacterium, yeast and aGram-negative bacterium such as a bacterium belonging to the genusPseudomonas (Japanese Published Examined Patent Application No.56852/82) and a process for producing a bran pickling composition bycollecting aromatic components of bran pickles by steam distillation andthen adding the collected components to the flavoring material (JapanesePublished Examined Patent Application No. 50697/83), but neither of themis effective for producing a clear bran pickles flavoring solution.

DISCLOSURE OF THE INVENTION

[0010] A need exists for a bran pickles flavoring solution useful formaking good bran pickles, a clear bran pickles flavoring solution andestablishment of processes for producing said flavoring solutions.

[0011] The present inventors have studied an industrial process forproducing a bran pickles flavoring solution which can easily impart topickles a mature flavor peculiar to bran pickles, specifically, aflavoring substance necessary for a bran pickles flavoring solution. Asa result, the present inventors have found that propionic acid andγ-dodecalactone and/or γ-dodecelactone play an important role as branpickles flavoring components, and after further studies, they havecompleted the present invention.

[0012] The present invention relates to the following (1)-(30).Hereinafter the following microorganisms are abbreviated as follows:

[0013] A microorganism having the ability to produce lactic acid; alactic acid-producing bacterium.

[0014] A microorganism having the ability to produce propionic acid; apropionic acid-producing bacterium.

[0015] A microorganism having the ability to produce γ-dodecalactoneand/or γ-dodecelactone; a γ-DD lactone-producing microorganism.

[0016] (1) A process for producing a bran pickles flavoring solution,which comprises culturing a lactic acid-producing bacterium, a propionicacid-producing bacterium and a γ-DD lactone-producing microorganism in abran suspension or a dispersed solution of treated rice bran(hereinafter referred to as a treated bran suspension) to form propionicacid and γ-dodecalactone and/or γ-dodecelactone in the culture, saidbran pickles flavoring solution comprising said culture.

[0017] (2) The process according to (1), wherein the culturing of themicroorganisms is carried out by culturing two microorganismsrespectively belonging to two groups arbitrarily selected from the threegroups of lactic acid-producing bacteria, propionic acid-producingbacteria and γ-DD lactone-producing microorganisms in one bransuspension or treated bran suspension and separately culturing amicroorganism belonging to the remaining group in another bransuspension or treated bran suspension, or by culturing separately threemicroorganisms respectively belonging to said three groups in a bransuspension or a treated bran suspension, and then the obtained culturesare mixed.

[0018] (3) The process according to (1), wherein the lacticacid-producing bacterium and the propionic acid-producing bacterium arecultured under anaerobic conditions and the γ-DD lactone-producingmicroorganism is cultured under aerobic conditions.

[0019] (4) The process according to (1), wherein the culturing of themicroorganisms is carried out by culturing the γ-DD lactone-producingmicroorganism in the culture of the lactic acid-producing bacterium, andthen culturing the propionic acid-producing bacterium in the obtainedculture.

[0020] (5) The process according to (1), wherein the culturing of themicroorganisms is carried out by culturing the propionic acid-producingbacterium in the culture of the lactic acid-producing bacterium, andseparately culturing the γ-DD lactone-producing microorganism in theculture of the lactic acid-producing bacterium, and then the obtainedcultures are mixed.

[0021] (6) The process according to (1), wherein the culturing of theγ-DD lactone-producing microorganism is carried out by culturing onekind of γ-DD lactone-producing microorganism under aerobic conditionsand under anaerobic conditions separately, or by culturing one kind ofγ-DD lactone-producing microorganism under aerobic conditions andculturing another kind of γ-DD lactone-producing microorganism underanaerobic conditions.

[0022] (7) The process according to any of (1)-(6), wherein the γ-DDlactone-producing microorganism is yeast.

[0023] (8) The process according to (7), wherein the yeast belongs tothe genus Saccharomyces, Kluyveromyces or Zygosaccharomyces.

[0024] (9) The process according to any of (1)-(6), wherein the γ-DDlactone-producing microorganism is a bacterium belonging to the genusCorynebacterium.

[0025] (10) The process according to (9), wherein the bacteriumbelonging to the genus Corynebacterium is Corynebacterium sp. NK-1 (FERMBP-6329).

[0026] (11) The process according to any of (1)-(5), wherein the lacticacid-producing bacterium belongs to the genus Lactobacillus,Pediococcus, Leuconostoc, Streptococcus, Enterococcus orBifidobacterium.

[0027] (12) The process according to any of (1)-(5), wherein thepropionic acid-producing bacterium belongs to the genusPropionibacterium.

[0028] (13) The process according to (1), wherein the treated rice branis prepared by treating rice bran with hydrolase.

[0029] (14) A bran pickles flavoring solution which is obtained by theprocess of any of (1)-(13).

[0030] (15) The bran pickles flavoring solution according to (14), whichcontains propionic acid in an amount of 500 ppm or more andγ-dodecalactone and/or γ-dodecelactone in an amount of 100 ppm or moreas bran pickles flavoring components.

[0031] (16) A bran pickles flavoring solution which contains propionicacid in an amount of 500 ppm or more and γ-dodecalactone and/orγ-dodecelactone in an amount of 100 ppm or more as bran picklesflavoring components.

[0032] (17) A pickling solution for making pickles having a flavor offoods pickled in rice bran paste (hereinafter referred to as a branpickling solution) which contains 50-3000 ppm propionic acid and 10-300ppm γ-dodecalactone and/or γ-dodecelactone as bran pickles flavoringcomponents.

[0033] (18) A process for producing a lactone, which comprises culturinga microorganism belonging to the genus Corynebacterium and having theability to produce at least one γ-lactone selected from the groupconsisting of γ-dodecalactone, γ-dodecelactone and γ-decalactone in amedium containing at least one fatty acid selected from the groupconsisting of oleic acid, linoleic acid and ricinoleic acid to form atleast one lactone selected from the group consisting of γ-dodecalactone,γ-dodecelactone and γ-decalactone in the culture, and then recoveringthe formed lactone from the culture.

[0034] (19) The process according to (18), wherein the microorganism isCorynebacterium sp. NK-1 (FERM BP-6329).

[0035] (20) Corynebacterium sp. NK-1 (FERM BP-6329).

[0036] (21) A clear bran pickles flavoring solution obtained by mixing:

[0037] a) a separated solution obtained by subjecting the bran picklesflavoring solution of any of (14)-(16) to solid-liquid separation, or

[0038] b) a separated solution obtained by culturing a lacticacid-producing bacterium and a propionic acid-producing bacterium in abran suspension or a treated bran suspension, and then subjecting theobtained culture to solid-liquid separation; with

[0039] c) an extract obtained by culturing a lactic acid-producingbacterium and a propionic acid-producing bacterium in a bran suspensionor a treated bran suspension, subjecting the obtained culture tosolid-liquid separation, and then extracting the obtained residue withan acidic aqueous solution of ethanol, or

[0040] d) an extract obtained by culturing a γ-DD lactone-producingmicroorganism, subjecting the obtained culture to solid-liquidseparation, and then extracting the obtained residue with an acidicaqueous solution of ethanol.

[0041] (22) A clear bran pickles flavoring solution which containspropionic acid in an amount of 500 ppm or more and γ-dodecalactoneand/or γ-dodecelactone in an amount of 20 ppm or more as bran picklesflavoring components, and which has the turbidity of 0.4 or less at 660nm when diluted 10 times with water.

[0042] (23) A clear bran pickling solution which contains 50-2000 ppmpropionic acid and 1-150 ppm γ-dodecalactone and/or γ-dodecelactone asbran pickles flavoring components, and which has the turbidity of 0.4 orless at 660 nm.

[0043] (24) A process for producing a lactone-containing solution, whichcomprises subjecting a culture of a microorganism having the ability toproduce a lactone to solid-liquid separation, recovering the residue,and then extracting the lactone from said residue with an acidic aqueoussolution of ethanol. Herein the term lactone means all lactones such asγ-lactone and δ-lactone.

[0044] (25) The process according to (21) or (24), wherein the acidicaqueous solution of ethanol is in the pH range of 1.0-6.5.

[0045] (26) The process according to (21), (24) or (25), wherein theacidic aqueous solution of ethanol contains ethanol in an amount of 10vol % or more.

[0046] (27) A process for producing pickles, which comprises picklingfoods in the bran pickles flavoring solution or the bran picklingsolution of any of (14)-(17) and (21)-(23).

[0047] (28) Pickles obtained by the process of (27).

[0048] (29) A seed strain for rice bran paste comprising a bacteriumbelonging to the genus Corynebacterium.

[0049] (30) A process for producing rice bran paste containingγ-dodecalactone and/or γ-dodecelactone, which comprises adding the seedstrain for rice bran paste of (29) to rice bran paste.

[0050] The present invention is described below in detail.

[0051] Described first are a process for producing a novel bran picklesflavoring solution containing propionic acid and γ-dodecalactone and/orγ-dodecelactone, which comprises culturing a lactic acid-producingbacterium, a propionic acid-producing bacterium and a γ-DDlactone-producing microorganism in a bran suspension or a treated bransuspension; a novel bran pickles flavoring solution comprising theobtained culture; and a novel γ-DD lactone-producing microorganism,Corynebacterium sp. NK-1.

[0052] As the lactic acid-producing bacterium, any microorganism whichhas the ability to produce lactic acid and is applicable to foods can beused. For example, bacteria belonging to the genus Lactobacillus,Pediococcus, Leuconostoc, Streptococcus, Enterococcus or Bifidobacteriumcan be used. The preferred bacteria are those belonging to the speciesLactobacillus plantarum, Pediococcus pentosaceus, Leuconostocmesenteroides, Streptococcus lactis, Streptococcus thermophilus,Streptococcus cremoris, Enterococcus faecalis and Bifidobacteriumbifidum. Particularly preferred are those of the species Lactobacillusplantarum and Enterococcus faecalis.

[0053] As the propionic acid-producing bacterium, any microorganismwhich has the ability to produce propionic acid and is applicable tofoods can be used. Preferred are bacteria belonging to the genusPropionibacterium used for producing cheese, etc., for example, bacteriabelonging to the species Propionibacterium freudenreichii andPropionibacterium thoenii.

[0054] As the γ-DD lactone-producing microorganism, any microorganismwhich has the ability to produce γ-dodecalactone and/or γ-dodecelactoneand is applicable to foods can be used. Preferred are yeast and bacteriabelonging to the genus Corynebacterium.

[0055] Examples of the γ-DD lactone-producing yeast are those belongingto the genus Saccharomyces, Kluyveromyces or Zygosaccharomyces.Preferred are yeasts belonging to the species Saccharomyces cerevisiae,Saccharomyces uvarum, Saccharomyces exigus, Kluyveromyces lactis,Kluyveromyces marxianus, Kluyveromyces thermotolerans, Zygosaccharomycesbailii and Zygosaccharomyces florentinus.

[0056] As the bacterium belonging to the genus Corynebacterium,Corynebacterium sp. NK-1 is preferably used.

[0057] Corynebacterium sp. NK-1 was isolated from bran paste. Themicrobiological properties of this strain and the basis foridentification are described below.

[0058] (A) Morphological Characteristics

[0059] 1) Morphology of cells: Short rod

[0060] Size: 1.0-1.2×0.8-1.0 μm

[0061] 2) Polymorphism of cells: Not observed

[0062] 3) Motility: Not observed

[0063] 4) Sporulation: Not observed

[0064] (B) Cultural Characteristics

[0065] The cultural characteristics of the strain when cultured on abouillon-agar plate medium and in a bouillon-liquid medium are shownbelow.

[0066] 1) Culturing on a bouillon-agar plate medium (1-2 days ofculturing)

[0067] a) Growth: Abundant

[0068] b) Color: Cream

[0069] c) Gloss: Observed

[0070] d) Diffusible pigments: Negative

[0071] 2) Culturing in a bouillon-liquid medium (1-2 days of culturing)

[0072] a) Growth on the surface: None

[0073] b) Turbidity: Positive

[0074] 3) Stab culture in a bouillon-gelatin medium

[0075] a) Growth: Abundant

[0076] b) Liquefaction of gelatin: Positive

[0077] 4) Litmus-milk reaction

[0078] a) Reaction: Alkali

[0079] b) Coagulation: Negative

[0080] c) Liquefaction: Negative

[0081] (C) Physiological Properties

[0082] 1) Gram staining: Positive

[0083] 2) Nitrate reduction: Positive in a bouillon medium

[0084] Negative in a succinic acid medium

[0085] 3) Denitrification reaction: Negative

[0086] 4) MR test: Negative

[0087] 5) VP test: Negative

[0088] 6) Indole production: Negative

[0089] 7) Hydrogen sulfide production: Positive

[0090] 8) Hydrolysis of starch: Negative

[0091] 9) Utilization of citric acid: Positive in Koser's medium

[0092] 10) Utilization of inorganic nitrogen sources

[0093] a) Nitrates: Positive

[0094] b) Ammonium salts: Positive

[0095] 11) Pigment production: None

[0096] 12) Urease: Positive

[0097] 13) Oxidase: Negative

[0098] 14) Catalase: Positive

[0099] 15) Growth range

[0100] a) pH range for growth: pH 5-11 (optimum pH: around pH 6)

[0101] b) Temperature range for growth: 11-45° C. (optimum temperature:around 29° C.)

[0102] 16) Attitude toward oxygen: Aerobic

[0103] 17) O-F test: Oxidative

[0104] 18) Acid production (aerobic conditions)

[0105] Growth was not observed by stationary culture. The resultsobtained by shaking culture are shown below.

[0106] +: Produced −: Not produced

[0107] a) L-Arabinose: −

[0108] b) D-Xylose: −

[0109] c) D-Glucose: +

[0110] d) D-Mannose: −

[0111] e) D-Fructose: +

[0112] f) D-Galactose: −

[0113] g) Maltose: −

[0114] h) Sucrose: −

[0115] i) Lactose: −

[0116] j) Trehalose: −

[0117] k) D-Sorbitol: −

[0118] l) D-Mannitol: −

[0119] m) Inositol: −

[0120] n) Glycerin: −

[0121] o) Starch: −

[0122] (D) Chemotaxonomic Properties

[0123] 1) DNA base composition (G+C mol %): 69.3

[0124] 2) Cellular lipids

[0125] Major quinone: MK-9 (H2)

[0126] Major fatty acid: C16:0

[0127] Mycolic acid: Coryneform

[0128] 3) Diamino acid composition of cell wall peptidoglycan: meso-A2pm

[0129] (E) Other Properties

[0130] The base sequence of 16S ribosome RNA (16S rRNA) gene is shown bySEQ ID NO: 1 in the Sequence Listing.

[0131] Taxonomical studies were made on the strain based on the abovemicrobiological properties referring to the descriptions in Bergey'sManual of Systematic Bacteriology, vol. 2 (1986), whereby the strain waspresumed to be a bacterium related to the genus Corynebacterium.Further, the base sequence of 16S rRNA was determined and moleculargenealogical analysis was carried out on the base sequence of 16S rRNAby the neighbor joining method using the base sequences ofmicroorganisms of the genus Corynebacterium and its related genera asthe reference sequences. As a result, the strain was classified in thegenus Corynebacterium.

[0132] The strain was thus identified as a bacterium belonging to thegenus Corynebacterium and was named Corynebacterium sp. NK-1.

[0133] This strain was deposited with the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (1-3, Higashi1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Apr. 23, 1998 withaccession number FERM BP-6329.

[0134] In the process for producing a bran pickles flavoring solutionaccording to the present invention, culturing of the lacticacid-producing bacterium, the propionic acid-producing bacterium and theγ-DD lactone-producing microorganism may be carried out by inoculatingthese microorganisms together in a bran suspension or a treated bransuspension as a medium, or by culturing first the lactic acid-producingbacterium and then the propionic acid-producing bacterium or the γ-DDlactone-producing microorganism, and subsequently culturing theremaining microorganism.

[0135] Alternatively, each of the above three microorganisms may becultured individually, or any one of the above microorganisms and theother two microorganisms may be cultured separately in a bran suspensionor a treated bran suspension, and after the culturing, the obtainedcultures may be mixed.

[0136] Further, it is possible to employ two or more kinds ofmicroorganisms which can produce the same substance. The order ofculturing, the combination of microorganisms and the kind of culturingcan be arbitrarily selected.

[0137] When the γ-DD lactone-producing microorganism is cultured underaerobic conditions, γ-dodecalactone and/or γ-dodecelactone is produced.On the other hand, when it is cultured under anaerobic conditions,production of γ-dodecalactone and/or γ-dodecelactone is suppressed, butthe resulting culture has a good flavor. Thus, a culture having apreferable flavor can be obtained by culturing the γ-DDlactone-producing microorganism under aerobic conditions and anaerobicconditions, respectively, and then mixing the obtained cultures.

[0138] As the medium of the present invention, a suspension which isprepared by dispersing rice bran or treated rice bran in water at aconcentration of 1-70 vol % may be employed.

[0139] As the rice bran, not only fresh rice bran, but also roasted ricebran and steamed rice bran can be used.

[0140] Examples of the treated rice bran include treated mattersobtained by decomposing components of rice bran with various enzymes.Suitable enzymes include hydrolase enzymes such as a lipid-hydrolyzingenzyme (e.g. lipase), a protein-hydrolyzing enzyme (e.g. protease andpeptidase) and a starch-hydrolyzing enzyme (e.g. amylase andglucoamylase).

[0141] The enzymatic treatment is carried out by adding an enzyme to abran suspension and subjecting the mixture to reaction at 30-60° C. for1-20 hours.

[0142] In the above culturing and enzymatic treatment of rice bran,vegetables or extracts thereof, marine products or extracts thereof,yeast extract, protein hydrolysate, starch hydrolysate, glucose, etc.may be added, if necessary.

[0143] Propionic acid-producing bacteria require lactic acid to producepropionic acid. Therefore, when the propionic acid-producing bacteriumemployed can not produce enough lactic acid, it is necessary that lacticacid be contained in the medium. In cases where the propionicacid-producing bacterium which does not produce enough lactic acid iscultured in a culture broth of the lactic acid-producing bacterium or iscultured together with the lactic acid-producing bacterium, there is noneed to add lactic acid to the medium. However, in cases where lacticacid does not exist or is not produced in a medium, lactic acid must beadded to the medium.

[0144] When the γ-DD lactone-producing microorganism is yeast,precursors of γ-dodecalactone and/or γ-dodecelactone are required. Thus,it is necessary to add precursors of γ-dodecalactone and/orγ-dodecelactone such as 10-hydroxyoctadecanoic acid and10-hydroxyoctadecenoic acid to the medium. In cases where the γ-DDlactone-producing microorganism is cultured in a culture broth of thelactic acid-producing bacterium or is cultured together with the lacticacid-producing bacterium, precursors of γ-dodecalactone and/orγ-dodecelactone are formed from oleic acid and linoleic acid in themedium by the action of the lactic acid-producing bacterium, and sothere is no need to add said precursors to the medium.

[0145] When the γ-DD lactone-producing microorganism is a bacteriumbelonging to the genus Corynebacterium, it is necessary that oleic acidand linoleic acid be contained in the medium. However, as rice brancontains these fatty acids, there is no need for addition thereof. Byculturing said bacterium in a bran suspension or treated rice bran,γ-dodecalactone and/or γ-dodecelactone can be formed and accumulated inthe culture.

[0146] Culturing of the three kinds of microorganisms is carried outusually at 10-37° C. and completed in 1-20 days, whereby propionic acidand γ-dodecalactone and/or γ-dodecelactone can be formed and accumulatedin the culture.

[0147] Culturing of the lactic acid-producing bacterium and thepropionic acid-producing bacterium is preferably carried out underanaerobic conditions, for example, by stationary culture. Culturing ofthe yeast and the bacterium belonging to the genus Corynebacterium ispreferably carried out under aerobic conditions, for example, by shakingculture or submerged spinner culture under aeration.

[0148] The culture obtained by the culturing is used, as such, as a branpickles flavoring solution. As the bran pickles flavoring solution,those which contain, as flavoring components, propionic acid in anamount of 500 ppm or more, preferably 1000 ppm or more, andγ-dodecalactone and/or γ-dodecelactone in an amount of 100 ppm or more,preferably 250 ppm or more are desirable in respect of bran picklesflavor. The bran pickles flavoring solutions which further contain0.1-10.0 wt % lactic acid and 3.0-20.0 wt % sodium chloride are moredesirable in respect of bran pickles flavor. It is preferred that sodiumchloride is contained at the above concentration also from thestandpoint of preservation.

[0149] The bran pickles flavoring solution, as such, can be used as abran pickling solution, or may be diluted with water, etc. to prepare abran pickling solution containing 50-3000 ppm propionic acid and 10-300ppm γ-dodecalactone and/or γ-dodecelactone. The bran pickling solutionmay further contain additives such as lactic acid, sodium chloride andsodium glutamate.

[0150] The clear bran pickles flavoring solution of the presentinvention is described below.

[0151] The clear bran pickles flavoring solution of the presentinvention is obtained by mixing the following separated solution withthe following extract.

[0152] Examples of the separated solution include a separated solutionobtained by subjecting the above bran pickles flavoring solution tosolid-liquid separation, and a separated solution obtained by culturingthe lactic acid-producing bacterium and the propionic acid-producingbacterium in a bran suspension or a treated bran suspension and thensubjecting the obtained culture to solid-liquid separation.

[0153] Examples of the extract include an extract obtained by subjectingthe above bran pickles flavoring solution to solid-liquid separation andthen extracting the obtained residue with an acidic aqueous solution ofethanol, and an extract obtained by culturing the γ-DD lactone-producingmicroorganism described below in a medium, subjecting the obtainedculture to solid-liquid separation, and then extracting the obtainedresidue with an acidic aqueous solution of ethanol.

[0154] The separated solution and the residue can be obtained bysubjecting the bran pickles flavoring solution or the culture tosolid-liquid separation. The solid-liquid separation can be effected byany conventional method, for example, methods using a filter press, arotary filter, a centrifuge, a dynamic filter, etc. Extraction ofγ-dodecalactone and/or γ-dodecelactone from the residue is carried outrepeatedly with an acidic aqueous solution of ethanol, andγ-dodecalactone and/or γ-dodecelactone can be selectively extracted fromthe residue.

[0155] The pH of the acidic aqueous solution of ethanol for use is1.0-6.5, preferably 2.0-6.0 and the concentration of ethanol in thesolution is 10 vol % or more, preferably 30 vol % or more. The pHadjustment is usually carried out by using lactic acid, hydrochloricacid or a culture of a lactic acid-producing bacterium. Extraction oflipids such as fatty acids in the culture can be suppressed by makingthe aqueous solution of ethanol acidic. By suppression of extraction ofsaid lipids, the cause of turbidity at the time of preparing a branpickles flavoring solution can be reduced remarkably, which enables theproduction of a good clear bran pickles flavoring solution.

[0156] Described below is a method of culturing a γ-DD lactone-producingmicroorganism in order to produce the clear bran pickles flavoringsolution.

[0157] As the γ-DD lactone-producing microorganism, any microorganismwhich has the ability to produce γ-dodecalactone and/or γ-dodecelactonecan be used. Any of the γ-DD lactone-producing microorganisms used forthe production of the above bran pickles flavoring solution may beemployed.

[0158] As the medium, any natural or synthetic medium which containsoleic acid and linoleic acid, and also carbon sources, nitrogen sources,inorganic salts, etc. capable of being assimilated by thelactone-producing microorganism can be used as well as a bran suspensionand treated rice bran.

[0159] When the γ-DD lactone-producing microorganism is yeast,precursors of γ-dodecalactone and/or γ-dodecelactone are required. Thus,it is necessary to add said precursors, for example,10-hydroxyoctadecanoic acid and 10-hydroxyoctadecenoic acid to themedium.

[0160] In cases where the γ-DD lactone-producing microorganism iscultured in a culture broth of the lactic acid-producing bacterium or iscultured together with the lactic acid-producing bacterium, precursorsof γ-dodecalactone and/or γ-dodecelactone are formed from oleic acid andlinoleic acid in the medium by the action of the lactic acid-producingbacterium, and so there is no need to add said precursors to the medium.

[0161] Examples of the carbon sources include carbohydrates such asglucose, fructose, sucrose, molasses, starch and starch hydrolysate,organic acids such as acetic acid and propionic acid, and alcohols suchas ethanol and propanol.

[0162] Examples of the nitrogen sources include ammonia, ammonium saltsof inorganic or organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate and ammonium phosphate, and other compoundscontaining nitrogen, as well as peptone, meat extract, yeast extract,corn steep liquor, casein hydrolysate, soybean cake, soybean cakehydrolysate, and various fermented cells and digested matters thereof.Examples of the inorganic substances include potassiumdihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate and calcium carbonate.

[0163] Preferred sources of fatty acids to be supplied to the mediuminclude rice bran, hydrolase-treated rice bran, a product of hydrolysisof food fat containing linoleic acid and oleic acid such as animal fat,vegetable fat or milk fat, and a mixture obtained by adding to saidhydrolysis product a food protein such as milk protein or vegetableprotein at the rate of 0.1-1.0 by weight.

[0164] The culturing is usually carried out under aerobic conditions,for example, by shaking culture or submerged spinner culture underaeration, at pH 3-9 at 15-40° C. for 2-7 days.

[0165] The clear bran pickles flavoring solution obtained by the aboveprocess contains propionic acid in an amount of 500 ppm or more,preferably 1000 ppm or more, and γ-dodecalactone and/or γ-dodecelactonein an amount of 20 ppm or more, preferably 40 ppm or more, as flavoringcomponents, and has the turbidity of 0.4 or less, preferably 0.2 or lessat 660 nm when diluted 10 times with water.

[0166] The clear bran pickles flavoring solution, as such, can be usedas a clear bran pickling solution, or if necessary, may be diluted withwater, etc. to prepare a clear bran pickling solution containing 50-2000ppm propionic acid and 1-150 ppm γ-dodecalactone and/or γ-dodecelactone.The clear bran pickling solution may further contain additives such aslactic acid, sodium chloride and sodium glutamate.

[0167] The bran pickles flavoring solution and the bran picklingsolution, whether clear or not, may additionally comprise, as may beappropriate, additives such as a seasoning (e.g. sodium glutamate),starch hydrolysate, a sugar (e.g. glucose and sucrose), proteinhydrolysate, yeast extract, vegetable extract, marine product extract,and an organic acid (e.g. lactic acid, acetic acid and succinic acid),as well as spice such as red pepper, Japanese pepper and citron.

[0168] Pickles having a flavor of good bran pickles can be obtained bypickling foods such as vegetables, seafoods and meat in the branpickling solution, whether clear or not, or sprinkling the bran picklingsolution over foods, etc.

[0169] Described below is a novel process for producing at least oneγ-lactone selected from the group consisting of γ-dodecalactone,γ-dodecelactone and γ-decalactone (hereinafter collectively referred toas r-lactones) by the use of a bacterium belonging to the genusCorynebacterium.

[0170] As the bacterium belonging to the genus Corynebacterium of thepresent invention, any bacterium which has the ability to formγ-dodecalactone from oleic acid, γ-dodecelactone from linoleic acid orγ-decalactone from ricinoleic acid can be used. An example of such astrain is Corynebacterium sp. NK-1 described above.

[0171] As for the medium and the method of culturing, those used forculturing the above γ-DD lactone-producing microorganism are applicableexcept that addition of ricinoleic acid to the medium is necessary.

[0172] Recovery of γ-lactones from the culture obtained by culturing iscarried out, for example, by column chromatography.

[0173] Described below is a method of extracting a lactone from afermentation broth obtained by culturing a lactone-producingmicroorganism.

[0174] As the lactone-producing microorganism, any microorganism havingthe ability to produce at least one of the γ-lactones and δ-lactones canbe used. Useful microorganisms include the γ-DD lactone-producingmicroorganisms used for producing the above bran pickles flavoringsolution. As the method of extracting a lactone from the culture, thesame method as that used for producing the above clear bran picklesflavoring solution is applicable.

[0175] That is, the residue is obtained by subjecting the culture tosolid-liquid separation. The solid-liquid separation can be effected byany of the above methods. A lactone can be selectively extracted fromthe residue by repeating extraction using an acidic aqueous solution ofethanol.

[0176] The pH of the acidic aqueous solution of ethanol for use is1.0-6.5, preferably 2.0-6.0 and the concentration of ethanol in thesolution is 10 vol % or more, preferably 30 vol % or more. The pHadjustment is usually carried out by using lactic acid or hydrochloricacid. Extraction of lipids such as fatty acids in the culture can besuppressed by making the aqueous solution of ethanol acidic.

[0177] The seed strain for rice bran paste of the present invention isdescribed below.

[0178] The seed strain for rice bran paste is characterized in that itcomprises a bacterium belonging to the genus Corynebacterium which hasthe ability to produce γ-dodecalactone and/or γ-dodecelactone.

[0179] Usually in rice bran paste exist lactic acid bacteria, and it isknown that propionic acid is accumulated in rice bran paste even in theabsence of a propionic acid bacterium. Therefore, the seed strain forrice bran paste of the present invention only needs to contain, as anessential component, a bacterium belonging to the genus Corynebacteriumwhich is capable of producing γ-dodecalactone and/or γ-dodecelactone. Ifnecessary, a lactic acid-producing bacterium or a propionicacid-producing bacterium may be added.

[0180] The bacterium belonging to the genus Corynebacterium, the lacticacid bacterium and the propionic acid bacterium useful for the seedstrain are the same as those useful in the above process for producingthe bran pickles flavoring solution of the present invention.

[0181] Examples of the rice bran paste to which the above seed strain isadded include fresh rice bran and a mixture of fresh rice bran androasted rice bran or steamed rice bran, which may comprise vegetables orextracts thereof, marine products or extracts thereof, yeast extract,protein hydrolysate, etc.

[0182] Addition of the above seed strain to such bran paste causesfermentation to form propionic acid and γ-dodecalctone and/orγ-dodecelactone in the bran paste, whereby bran paste which is usefulfor making pickles having an excellent flavor of bran pickles can beobtained.

BRIEF DESCRIPTION OF THE DRAWINGS

[0183]FIG. 1 is a graph showing the concentration of ethanol in thesolution used for extraction and the concentration of extractedγ-lactone.

[0184]FIG. 2 is a graph showing the pH of the solution used forextraction and the concentrations of extracted γ-lactone and oleic acid.

[0185]FIG. 3 is a graph showing the pH of the solution used forextraction and the turbidity of the bran pickles flavoring solution.

BEST MODES FOR CARRYING OUT THE INVENTION

[0186] Certain specific embodiments of the present invention areillustrated in the following examples. In the examples, % means wt %unless otherwise specified.

EXAMPLE 1

[0187] A dispersed solution prepared by mixing rice bran (Tajimaya) withwater at a concentration of 10% (referred to as a 10% bran suspension)was heated to 85° C. and then cooled to 50° C. To the bran suspensionwere added protease (Novo Nordisk A/S), peptidase (Novo Nordisk A/S),amylase (Nagase Seikagaku Co., Ltd.), glucoamylase (Nagase SeikagakuCo., Ltd.) and lipase (Amano Pharmaceutical Co., Ltd.) which aresuitable for use in food processing in an amount of 0.1% based on therice bran, followed by reaction at 50° C. for 3 hours. After thereaction was completed, the reaction mixture was sterilized by heatingat 120° C. for 15 minutes. Lactobacillus plantarum was inoculatedtherein as a lactic acid bacterium, followed by stationary culture at25° C. for 2 days. Then, Saccharomyces cerevisiae was inoculated thereinas a γ-DD lactone-producing yeast, followed by spinner culture underaeration at 25° C. for 4 days. After the culturing was completed,fermented lactic acid (90% lactic acid, Musashino Shoji Co., Ltd.) wasadded to the obtained culture in an amount of 2% based on said culture,and Propionibacterium freudenreichii was inoculated therein as apropionic acid-producing bacterium, followed by stationary culture at25° C. for 7 days.

[0188] After the culturing was completed, the obtained culture wassterilized by heating at 95° C. for 30 minutes. The amounts of propionicacid, γ-dodecalactone and γ-dodecelactone in said culture weredetermined. The amount of propionic acid was determined by capillaryelectrophoresis (HP 3D CE, Hewlett Packard). The amounts ofγ-dodecalactone and γ-dodecelactone were determined by means of a massspectrum detector (QP5000, Shimadzu Corporation) after extraction fromsaid culture with a pentane/ether mixture (1:1) using cyclohexanol as aninternal standard and separation by gas chromatography. As a result, itwas found that said culture contained 1.1% propionic acid, 0.11%γ-dodecalactone and 0.07% γ-dodecelactone, and also had a flavor ofmature bran pickles.

[0189] The obtained culture, which is a bran pickles flavoring solution,was diluted 10 times with water, and lactic acid, sodium chloride andsodium glutamate were added thereto to the concentrations of 0.5%, 2.5%and 0.62%, respectively, to prepare a pickling solution (solution of thepresent invention). In this pickling solution were pickled cucumberswhich had been previously pickled in a 6% aqueous solution of sodiumchloride at 5° C. for one day. Separately, a commercially available branpickles flavoring solution (hereinafter referred to as commercialsolution 1) was diluted according to the attached description, andcucumbers were pickled in the resulting pickling solution in the samemanner as above. After pickling at 5° C. for 2 days, the picklingsolution was removed and the pickled cucumbers were sliced. Evaluationof the sliced cucumbers was made in respect of mature flavor, branpickles flavor and desirability as bran pickles by 12 panelists eachhaving 7 points on each quality.

[0190] The results are shown in Table 1. TABLE 1 Bran picklesDesirability as Mature flavor flavor bran pickles Solution of the 5.11 ±1.45 5.00 ± 1.01 4.97 ± 1.11 present inv. Commercial 3.12 ± 1.16 2.89 ±1.21 2.16 ± 1.46 solution 1

[0191] As shown in Table 1, the solution of the present invention gavepickles superior to those obtained by using commercial solution 1 in allof the above qualities, i.e. mature flavor, bran pickles flavor anddesirability as bran pickles.

EXAMPLE 2

[0192] After a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, Enterococcusfaecalis was inoculated therein as a lactic acid bacterium, followed bystationary culture at 25° C. for 2 days. After the culturing wascompleted, the obtained culture was divided into two equal portions.Into one portion was inoculated Zygosaccharomyces bailii as a γ-DDlactone-producing yeast, followed by spinner culture under aeration for5 days. Into the other portion was inoculated Propionibacteriumfreudenreichii as a propionic acid-producing bacterium, followed bystationary culture for 5 days. After the culturing was completed, bothcultures were sterilized by heating at 95° C. for 30 minutes and sodiumchloride was added thereto in an amount of 5%, followed by mixing of thecultures. The obtained mixture had a flavor of mature bran pickles andcontained 1.2% propionic acid, 0.08% γ-dodecalactone and 0.09%γ-dodecelactone.

[0193] Cucumbers were pickled in an aqueous solution prepared bydiluting said mixture 10 times (solution of the present invention) andin a commercially available bran pickles flavoring solution (commercialsolution 1) according to the method described in Example 1, andevaluation of the pickled cucumbers was made in respect of the samequalities by 12 panelists each having 7 points on each quality.

[0194] The results are shown in Table 2. TABLE 2 Bran picklesDesirability as Mature flavor flavor bran pickles Solution of the 4.84 ±1.36 4.79 ± 1.21 5.00 ± 1.15 present inv. Commercial 2.67 ± 1.45 3.11 ±1.33 2.22 ± 1.51 solution 1

[0195] As shown in Table 2, the solution of the present invention gavepickles superior to those obtained by using commercial solution 1 in allof the evaluated qualities, i.e. mature flavor, bran pickles flavor anddesirability as bran pickles.

EXAMPLE 3

[0196] After a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, Lactobacillusplantarum was inoculated therein as a lactic acid-producing bacterium,followed by stationary culture at 25° C. for 2 days. Further,Propionibacterium freudenreichii was inoculated therein as a propionicacid-producing bacterium, followed by stationary culture at 25° C. for 7days. Separately, a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, and a γ-DDlactone-producing microorganism Corynebacterium sp. NK-1 was inoculatedtherein, followed by spinner culture under aeration for 5 days. Afterthe culturing was completed, both cultures were mixed. The obtainedmixture had a flavor of mature bran pickles and contained 0.7% propionicacid, 0.08% γ-dodecalactone and 0.06% γ-dodecelactone.

[0197] Cucumbers were pickled in an aqueous solution prepared bydiluting said mixture 10 times (solution of the present invention) andin a commercially available bran pickles flavoring solution (commercialsolution 1) according to the method described in Example 1, andevaluation of the pickled cucumbers was made in respect of the samequalities by 12 panelists each having 7 points on each quality.

[0198] The results are shown in Table 3. TABLE 3 Bran picklesDesirability as Mature flavor flavor bran pickles Solution of the 5.24 ±1.19 5.31 ± 1.00 5.02 ± 0.92 present inv. Commercial 2.97 ± 1.21 2.99 ±1.13 2.32 ± 1.52 solution 1

[0199] As shown in Table 3, the solution of the present invention gavepickles superior to those obtained by using commercial solution 1 in allof the evaluated qualities, i.e. mature flavor, bran pickles flavor anddesirability as bran pickles.

EXAMPLE 4

[0200] After a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, Lactobacillusplantarum was inoculated therein as a lactic acid-producing bacterium,followed by stationary culture at 25° C. for 2 days.

[0201] Separately, a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, andLactobacillus plantarum was inoculated therein as a lacticacid-producing bacterium, followed by stationary culture at 25° C. for 2days. Further, Propionibacterium freudenreichii was inoculated thereinas a propionic acid-producing bacterium, followed by stationary cultureat 25° C. for 7 days.

[0202] Another 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, andLactobacillus plantarum was inoculated therein as a lacticacid-producing bacterium, followed by stationary culture at 25° C. for 2days. Further, Saccharomyces cerevisiae was inoculated therein as a γ-DDlactone-producing microorganism, followed by stationary culture at 25°C. for 4 days.

[0203] Further, another 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, andCorynebacterium sp. NK-1 was inoculated therein as a γ-DDlactone-producing microorganism, followed by spinner culture underaeration for 5 days.

[0204] After the culturing was completed, the obtained four kinds ofcultures were sterilized by heating to 95° C. and mixed in equalamounts. The obtained mixture had a flavor of mature bran pickles andcontained 0.25% propionic acid, 0.04% γ-dodecalactone and 0.03%γ-dodecelactone.

[0205] Cucumbers were pickled in a solution prepared by diluting saidmixture 10 times (solution of the present invention) and in acommercially available bran pickles flavoring solution (commercialsolution 1) according to the method described in Example 1, andevaluation of the pickled cucumbers was made in respect of the samequalities by 12 panelists each having 7 points on each quality.

[0206] The results are shown in Table 4. TABLE 4 Bran picklesDesirability as Mature flavor flavor bran pickles Solution of the 4.62 ±1.00 4.56 ± 0.98 4.56 ± 1.43 present inv. Commercial 2.81 ± 1.36 2.67 ±1.11 2.09 ± 0.87 solution 1

[0207] As shown in Table 4, the solution of the present invention gavepickles superior to those obtained by using commercial solution 1 in allof the evaluated qualities, i.e. mature flavor, bran pickles flavor anddesirability as bran pickles.

EXAMPLE 5

[0208] After a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, Lactobacillusplantarum was inoculated therein as a lactic acid-producing bacterium,followed by stationary culture at 25° C. for 2 days.

[0209] Separately, a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, andLactobacillus plantarum was inoculated therein as a lacticacid-producing bacterium, followed by stationary culture at 25° C. for 2days. Further, Propionibacterium freudenreichii was inoculated thereinas a propionic acid-producing bacterium, followed by stationary cultureat 25° C. for 7 days.

[0210] Another 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, andEnterococcus faecalis was inoculated therein as a lactic acid-producingbacterium, followed by stationary culture at 25° C. for 2 days. Further,Zygosaccharomyces bailii was inoculated therein, followed by stationaryculture at 25° C. for 4 days.

[0211] Further, another 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, andEnterococcus faecalis was inoculated therein as a lactic acid-producingbacterium, followed by stationary culture at 25° C. for 2 days. Then,Zygosaccharomyces bailii was inoculated therein as a γ-DDlactone-producing microorganism, followed by spinner culture underaeration for 5 days.

[0212] After the culturing was completed, the obtained four kinds ofcultures were sterilized by heating to 95° C. and mixed in equalamounts. The obtained mixture had a flavor of mature bran pickles andcontained 0.20% propionic acid, 0.03% γ-dodecalactone and 0.02%γ-dodecelactone.

[0213] Cucumbers were pickled in a solution prepared by diluting saidmixture 10 times (solution of the present invention) and in acommercially available bran pickles flavoring solution (commercialsolution 1) according to the method described in Example 1, andevaluation of the pickled cucumbers was made in respect of the samequalities by 12 panelists each having 7 points on each quality.

[0214] The results are shown in Table 5. TABLE 5 Bran picklesDesirability as Mature flavor flavor bran pickles Solution of the 4.33 ±1.40 4.00 ± 0.89 4.19 ± 1.21 present inv. Commercial 2.45 ± 1.17 2.03 ±1.32 2.01 ± 1.31 solution 1

[0215] As shown in Table 5, the solution of the present invention gavepickles superior to those obtained by using the commercial solution inall of the evaluated qualities, i.e. mature flavor, bran pickles flavorand desirability as bran pickles.

EXAMPLE 6

[0216] To a 10% bran suspension was added castor oil to a concentrationof 1%, and the resulting mixture was treated with enzymes and sterilizedby heating in the same manner as in Example 1. Then, Corynebacterium sp.NK-1 was inoculated in the mixture, followed by spinner culture underaeration for 4 days.

[0217] As a result, a culture containing 692 ppm γ-dodecalactone, 243ppm γ-dodecelactone and 572 ppm γ-decalactone was obtained.

EXAMPLE 7

[0218] To a medium (0.1% yeast extract, 0.5% peptone, 2% skim milk)containing 4% corn oil was added lipase in an amount of 1% based on thecorn oil, followed by reaction at 37° C. for 10 hours to obtain corn oilhydrolysate. After the obtained corn oil hydrolysate was sterilized byheating, Corynebacterium sp. NK-1 was inoculated therein, followed byspinner culture under aeration for 5 days.

[0219] As a result, a culture containing 1850 ppm γ-dodecalactone and558 ppm γ-dodecelactone was obtained.

EXAMPLE 8

[0220] Bran paste (5 kg) comprising 50% rice bran, 47% water and 3%sodium chloride was stirred and then fermented at 15° C. for 3 weeks,during which 5 cucumbers were pickled in the bran paste every day andtaken out on the following day. The resulting fermented bran paste wasdivided into 2 portions, and one portion was inoculated with culturedcells of Corynebacterium sp. NK-1 in an amount of 0.1%. Then, eachportion was stirred and then fermented at 15° C. for 2 weeks, duringwhich 5 cucumbers were pickled in each bran paste every day and takenout on the following day. As a result, γ-dodecalactone andγ-dodecelactone were detected in a total amount of 32 ppm in thecell-inoculated bran paste, which had a good flavor, whileγ-dodecalactone or γ-dodecelactone was not detected in the bran pastewithout incubation of the cells.

EXAMPLE 9

[0221] Bran paste (2 kg) comprising 50% rice bran, 47% water and 3%sodium chloride was stirred and then fermented at 15° C. for 3 weeks,during which 5 cucumbers were pickled in the bran paste every day andtaken out on the following day. To the resulting fermented bran pastewas added the mixture obtained in Example 3 (a bran pickles flavoringsolution) in an amount of 10%, followed by mixing, and 5 cucumbers werepickled in the resulting bran paste. The pickled cucumbers had a goodflavor of bran pickles.

EXAMPLE 10

[0222] After a 10% bran suspension was treated with enzymes andsterilized by heating in the same manner as in Example 1, Lactobacillusplantarum was inoculated therein as a lactic acid-producing bacterium,followed by stationary culture at 25° C. for 2 days. Then, Saccharomycescerevisiae was inoculated therein as a γ-DD lactone-producing yeast,followed by spinner culture under aeration at 25° C. for 4 days.

[0223] Separately, a 2% bran suspension was treated with enzymes in thesame manner as in Example 1, and fermented lactic acid was added theretoin an amount of 2%. After the resulting mixture was sterilized byheating, Propionibacterium freudenreichii was inoculated therein as apropionic acid-producing bacterium, followed by stationary culture at25° C. for 6 days.

[0224] After the culturing was completed, both cultures were mixed. Theobtained mixture (solution of the present invention 1) had a flavor ofmature bran pickles and contained 3611 ppm propionic acid, 154 ppmγ-dodecalactone and 358 ppm γ-dodecelactone.

[0225] Said mixture (30 l) was filtered by means of a filter press pilot(Yabuta Kikai Co., Ltd.) to obtain the filtrate containing propionicacid. Then, 10 l of an aqueous solution containing 59% (v/v) ethanol (pH4.7) was circulated through the residue for extraction. The obtainedextract was mixed with the above filtrate to give a concentrated clearbran pickles flavoring solution (solution of the present invention 2)which had the turbidity of 0.021 at 660 nm and contained 2544 ppmpropionic acid, 25.2 ppm γ-dodecalactone and 60.1 ppm γ-dodecelactone.

[0226] Measurements were made on solutions of the present invention 1and 2 and commercially available bran pickles flavoring solutions(commercial solutions 1, 2 and 3) in respect of turbidity and content ofcomponents related to a bran pickles flavor. The turbidity is expressedin terms of the turbidity at 660 nm when each solution is diluted 10times with water, and the content of flavoring components is expressedin terms of the concentration in each solution undiluted.

[0227] The results are shown in Table 6. TABLE 6 γ-Dodeca- lactone +γ-dodece- Turbidity Propionic lactone Dilution (660 nm) acid (ppm) (ppm)Solution of 10-20 times 3.51 3611 512.0 the present inv. 1 Solution of10-20 times 0.001 2544 85.3 the present inv. 2 Commercial  7-10 times0.008 tr 1.4 solution 1 Commercial  7-10 times 9.41  422 tr solution 2Commercial 20-40 times 0.056 tr tr solution 3

[0228] Sensory evaluation was made on the cucumber pickles prepared byusing solutions of the present invention 1 and 2 and commercialsolutions 1, 2 and 3. Each of the solutions was diluted 10 times, andlactic acid, sodium chloride and sodium glutamate were added thereto tothe concentrations of 0.5%, 2.5% and 0.62%, respectively, to prepare apickling solution. In the obtained solution were pickled cucumbers whichhad been previously pickled in a 6% aqueous solution of sodium chlorideat 5° C. for one day. After pickling at 5° C. for 2 days, the picklingsolution was removed and the cucumbers were cut. Evaluation of the cutcucumbers was made in respect of mature flavor, bran pickles flavor anddesirability as bran pickles by 11 panelists each having 7 points oneach quality.

[0229] The results are shown in Table 7. TABLE 7 Bran picklesDesirability as Mature flavor flavor bran pickles Solution of the  5.12± 0.65*  5.21 ± 1.09*  4.50 ± 1.56* present inv. 1 Solution of the  5.09± 1.45*  5.36 ± 1.12*  5.27 ± 0.90* present inv. 2 Commercial 2.82 ±1.25 2.45 ± 1.13 2.91 ± 1.87 solution 1 Commercial 3.55 ± 1.13 3.09 ±1.51 2.27 ± 1.19 solution 2 Commercial 2.09 ± 1.30 2.00 ± 1.34 2.36 ±1.12 solution 3

[0230] As shown in Table 7, the solutions of the present invention gavepickles superior to those obtained by using the commercial solutions inall of the above qualities, i.e. mature flavor, bran pickles flavor anddesirability as bran pickles.

EXAMPLE 11

[0231] A pickling solution was prepared from solution of the presentinvention 2 obtained in Example 10 in the same manner as in Example 10,and cut cucumbers were pickled therein at 5° C. for 30 minutes. As aresult, lightly pickled cucumbers having a flavor of bran pickles wereobtained. Cucumber pickles prepared by sprinkling the above solutionover cut cucumbers also had a flavor of bran pickles.

EXAMPLE 12

[0232] A 10% bran suspension was treated with enzymes and sterilized byheating in the same manner as in Example 1, and Lactobacillus plantarumwas inoculated therein as a lactic acid-producing bacterium, followed bystationary culture at 25° C. for 3 days. Then, Saccharomyces cerevisiaewas inoculated therein as a γ-DD lactone-producing yeast, followed byspinner culture under aeration at 25° C. for 4 days to obtain a culturecontaining γ-dodecalactone and γ-dodecelactone in a total amount of 2872ppm. Separately, a 3% bran suspension was treated with enzymes in thesame manner as in Example 1, and fermented lactic acid was added theretoin an amount of 3%. After the resulting mixture was sterilized byheating, Propionibacterium freudenreichii was inoculated therein as apropionic acid-producing bacterium, followed by stationary culture at25° C. for 7 days to obtain a culture containing 2.51% propionic acid.Both cultures thus obtained were mixed, and lactic acid, sodium chlorideand sodium glutamate were added thereto to the concentrations of 0.5%,2.5% an 0.62%, respectively. From this mixture were prepared picklingsolutions containing propionic acid, γ-dodecalactone and γ-dodecelactoneat the concentrations shown in Table 8. In each of the picklingsolutions (adjusted to pH 4.5) were pickled cucumbers which had beenpreviously pickled in a 6% aqueous solution of sodium chloride at 5° C.for one day. After pickling at 5° C. for 2 days, the pickling solutionwas removed and the cucumbers were sliced. Evaluation of the slicedcucumbers was made in respect of bran pickles flavor and desirability asbran pickles by 9 panelists, in which each quality was evaluated into 4grades.

[0233] The bran pickles flavor was evaluated into the following 4grades: −, weak; +, rather strong; ++, strong; and +++, very strong. Thedesirability as bran pickles was evaluated into the following 4 grades:−, little desirable; +, rather desirable; ++, desirable; and +++, highlydesirable.

[0234] The average values of the evaluation are shown in Table 8. TABLE8 γ-Dodeca- lactone + γ- Propionic acid dodecelactone Bran picklesDesirability as (ppm) (ppm) flavor bran pickles 0 0 − − 0 100 − − 20 100− − 50 100 + + 100 100 ++ ++ 250 100 ++ ++ 500 100 ++ +++ 1000 100 ++++++ 2000 100 + +++ 3000 100 + ++ 4000 100 − ++ 500 0 − − 500 5.0 − − 50010.0 + + 500 25.0 ++ ++ 500 100.0 ++ +++ 500 200.0 +++ +++ 500 300.0++ + 500 400.0 − −

EXAMPLE 13

[0235] A culture of a propionic acid-producing bacterium prepared in thesame manner as in Example 12 (30 l) was filtered by means of a filterpress pilot (Yabuta Kikai Co., Ltd.) to obtain the filtrate containingpropionic acid.

[0236] Separately, a culture of a γ-DD lactone-producing yeast preparedin the same manner as in Example 12 (30 l) was filtered by means of afilter press pilot (Yabuta Kikai Co., Ltd.), and 10 l of an aqueoussolution containing 59% (v/v) ethanol (pH 4.7) was circulated throughthe residue for extraction to obtain a 1151 ppm lactone solution. Theobtained lactone solution was mixed with the above filtrate, and lacticacid, sodium chloride and sodium glutamate were added thereto to theconcentrations of 0.5%, 2.5% and 0.62%, respectively. From this mixturewere prepared pickling solutions containing propionic acid,γ-dodecalactone and γ-dodecelactone at the concentrations shown in Table9. In each of the pickling solutions (adjusted to pH 4.5) were pickledcucumbers which had been previously pickled in a 6% aqueous solution ofsodium chloride at 5° C. for one day. After pickling at 5° C. for 2days, the pickling solution was removed and the cucumbers were sliced.Evaluation of the sliced cucumbers was made in respect of bran picklesflavor and desirability as bran pickles by 14 panelists. The qualitieswere evaluated into 4 grades in the same manner as in Example 12.

[0237] The average values of the evaluation are shown in Table 9. TABLE9 γ-Dodeca- lactone + γ- Propionic acid dodecelactone Bran picklesDesirability as (ppm) (ppm) flavor bran pickles 0 0 − − 0 17.5 − − 2017.5 − − 50 17.5 + + 100 17.5 ++ ++ 250 17.5 ++ ++ 500 17.5 +++ +++ 100017.5 ++ +++ 2000 17.5 + ++ 3000 17.5 − + 360 17.5 ++ ++ 360 0 − − 3601.0 − − 360 2.0 + + 360 4.0 ++ ++ 360 17.5 +++ +++ 360 70.0 +++ +++ 360150.0 ++ + 360 200.0 + −

EXAMPLE 14

[0238] A culture containing 7-dodecalactone and γ-dodecelactone in atotal amount of 1540 ppm as bran pickles flavoring components wasfiltered by pressure, and a half portion of the residue was extractedwith 0-50 vol % aqueous solutions of ethanol. As a result,γ-dodecalactone and γ-dodecelactone were recovered when the ethanolconcentration was 10 vol % or higher, and the recovery yield wasremarkably enhanced by the use of a solution having the etherconcentration of 30 vol % or higher (refer to FIG. 1).

[0239] The other half portion of the residue was extracted with 35%aqueous solutions of ethanol varied in pH to measure the amount of fattyacids extracted. As a result, extraction of fatty acids was suppressedat pH 6.5 or lower (refer to FIG. 2).

[0240] Said extracts were respectively mixed with the filtrate obtainedby filtration to prepare clear bran pickles flavoring solutions. Each ofthe resulting solutions was diluted 10 times with water, and theturbidity was measured. As a result, it was demonstrated that when theaqueous solution of ethanol is acidic, specifically at pH 6.5 or lower,the turbidity of a bran pickles flavoring solution is low and a clearbran pickles flavoring solution having a good flavor can be prepared(refer to FIG. 3).

INDUSTRIAL APPLICABILITY

[0241] The present invention provides processes for producing thefollowing: a novel bran pickles flavoring solution, a novel clear branpickles flavoring solution, pickles made by pickling foods in saidflavoring solutions, lactones which are essential components of saidflavoring solutions, and rice bran paste containing said lactones.

1 1 1 1460 DNA Corynebacterium sp.NK-1 unsure (166) n is “a” or “g” or“c” or “t”. 1 ggttcaggac ggaacgctgg cggcgtgctt aacacatgca agtcgaacggaaaggccggg 60 tgcttgcacc cggtactcga gtggcgaacg ggtgagtaac acgtgggtgatctgccctgc 120 acttcgggat aagcctggga aactgggtct aataccggat aggacnaccggttggtactg 180 gtggtggaaa gtttttcggt gcaggatgag ctcgcggcct atcagcttgttggtggggta 240 atggcctacc aaggcggcga cgggtagccg gcctgagagg gtggacggccacattgggac 300 tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattgcacaatgggcgca 360 agcctgatgc agcgacgccg cgtgggggat gacggccttc gggttgtaaactcctttcaa 420 ccatgacgaa gcttttgtga cggtagtggt agaagaagca ccggctaactacgtgccagc 480 agccgcggta atacgtaggg tgcgagcgtt gtccggaatt actgggcgtaaagagctcgt 540 aggtggtttg tcgcgtcgtc tgtgaaattc cggggctcaa ctccgggcgtgcaggcgata 600 cgggcataac ttgagtgctg taggggagac tggaattcct ggtgtagcggtgaaatgcgc 660 agatatcagg aggaacaccg atggcgaagg caggtctctg ggcagtaactgacgctgagg 720 agcgaaagca tgggtagcga acaggattag ataccctggt agtccatgccgtaaacggtg 780 ggcgctaggt gtgggggtct tccacgactt ctgtgccgta gctaacgcattaagcgcccc 840 gcctggggag tacggccgca aggctaaaac tcaaaggaat tgacgggggcccgcacaagc 900 cgcggagcat gtggattaat tcgatgcaac gcgaagaacc ttacctgggcttgacatgta 960 ccggatcggc gcagagatgt gtcttccctt gtggtcggta tacaggtggtgcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaacccttgtcctg 1080 tgttgccagc acgttgtggt ggggactcac gggagactgc cggggttaactcggaggaag 1140 gtggggatga cgtcaaatca tcatgcccct tatgtccagg gcttcacacatgctacaatg 1200 gtcggtacag tgggttgcta caccgtgagg tggtgctaat ctcttaaagccggtctcagt 1260 tcggattgga gtctgcaact cgactccatg aagtcggagt cgctagtaatcgcagatcag 1320 caatgctgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcacgtcatgaaagt 1380 tggtaacacc cgaagccggt ggcccaaact cgttagggag ccgtcgaaggtgggatcggc 1440 gattggacga aatcctacaa 1460

1. A process for producing a flavoring solution for making pickleshaving a flavor of foods pickled in rice bran paste (hereinafterreferred to as a bran pickles flavoring solution), which comprisesculturing a microorganism having the ability to produce lactic acid, amicroorganism having the ability to produce propionic acid and amicroorganism having the ability to produce γ-dodecalactone and/orγ-dodecelactone in a dispersed solution of rice bran (hereinafterreferred to as a bran suspension) or a dispersed solution of treatedrice bran (hereinafter referred to as a treated bran suspension) to formpropionic acid and γ-dodecalactone and/or γ-dodecelactone in theculture, said bran pickles flavoring solution comprising said culture.2. The process according to claim 1, wherein the culturing of themicroorganisms is carried out by culturing two microorganismsrespectively belonging to two groups arbitrarily selected from the threegroups of microorganisms having the ability to produce lactic acid,microorganisms having the ability to produce propionic acid andmicroorganisms having the ability to produce γ-dodecalactone and/orγ-dodecelactone in one bran suspension or treated bran suspension andseparately culturing a microorganism belonging to the remaining group inanother bran suspension or treated bran suspension, or by culturingseparately three microorganisms respectively belonging to said threegroups in a bran suspension or a treated bran suspension, and then theobtained cultures are mixed.
 3. The process according to claim 1,wherein the microorganism having the ability to produce lactic acid andthe microorganism having the ability to produce propionic acid arecultured under anaerobic conditions, and the microorganism having theability to produce γ-dodecalactone and/or γ-dodecelactone is culturedunder aerobic conditions.
 4. The process according to claim 1, whereinthe culturing of the microorganisms is carried out by culturing themicroorganism having the ability to produce γ-dodecalactone and/orγ-dodecelactone in the culture of the microorganism having the abilityto produce lactic acid, and then culturing the microorganism having theability to produce propionic acid in the obtained culture.
 5. Theprocess according to claim 1, wherein the culturing of themicroorganisms is carried out by culturing the microorganism having theability to produce propionic acid in the culture of the microorganismhaving the ability to produce lactic acid, and separately culturing themicroorganism having the ability to produce γ-dodecalactone and/orγ-dodecelactone in the culture of the microorganism having the abilityto produce lactic acid, and then the obtained cultures are mixed.
 6. Theprocess according to claim 1, wherein the culturing of the microorganismhaving the ability to produce γ-dodecalactone and/or γ-dodecelactone iscarried out by culturing one kind of microorganism having the ability toproduce γ-dodecalactone and/or γ-dodecelactone under aerobic conditionsand under anaerobic conditions separately, or by culturing one kind ofmicroorganism having the ability to produce γ-dodecalactone and/orγ-dodecelactone under aerobic conditions and culturing another kind ofmicroorganism having the ability to produce γ-dodecalactone and/orγ-dodecelactone under anaerobic conditions.
 7. The process according toany of claims 1-6, wherein the microorganism having the ability toproduce γ-dodecalactone and/or γ-dodecelactone is yeast.
 8. The processaccording to claim 7, wherein the yeast belongs to the genusSaccharomyces, Kluyveromyces or Zygosaccharomyces.
 9. The processaccording to any of claims 1-6, wherein the microorganism having theability to produce γ-dodecalactone and/or γ-dodecelactone is a bacteriumbelonging to the genus Corynebacterium.
 10. The process according toclaim 9, wherein the bacterium belonging to the genus Corynebacterium isCorynebacterium sp. NK-1 (FERM BP-6329).
 11. The process according toany of claims 1-5, wherein the microorganism having the ability toproduce lactic acid is a bacterium belonging to the genus Lactobacillus,Pediococcus, Leuconostoc, Streptococcus, Enterococcus orBifidobacterium.
 12. The process according to any of claims 1-5, whereinthe microorganism having the ability to produce propionic acid is abacterium belonging to the genus Propionibacterium.
 13. The processaccording to claim 1, wherein the treated rice bran is prepared bytreating rice bran with hydrolase.
 14. A bran pickles flavoring solutionwhich is obtained by the process of any of claims 1-13.
 15. The branpickles flavoring solution according to claim 14, which containspropionic acid in an amount of 500 ppm or more and γ-dodecalactoneand/or γ-dodecelactone in an amount of 100 ppm or more as bran picklesflavoring components.
 16. A bran pickles flavoring solution whichcontains propionic acid in an amount of 500 ppm or more andγ-dodecalactone and/or γ-dodecelactone in an amount of 100 ppm or moreas bran pickles flavoring components.
 17. A pickling solution for makingpickles having a flavor of foods pickled in rice bran paste (hereinafterreferred to as a bran pickling solution) which contains 50-3000 ppmpropionic acid and 10-300 ppm 7-dodecalactone and/or γ-dodecelactone asbran pickles flavoring components.
 18. A process for producing alactone, which comprises culturing a microorganism belonging to thegenus Corynebacterium and having the ability to produce at least oneγ-lactone selected from the group consisting of γ-dodecalactone,γ-dodecelactone and γ-decalactone in a medium containing at least onefatty acid selected from the group consisting of oleic acid, linoleicacid and ricinoleic acid to form at least one lactone selected from thegroup consisting of γ-dodecalactone, γ-dodecelactone and γ-decalactonein the culture, and then recovering the formed lactone from the culture.19. The process according to claim 18, wherein the microorganism isCorynebacterium sp. NK-1 (FERM BP-6329).
 20. Corynebacterium sp. NK-1(FERM BP-6329).
 21. A clear bran pickles flavoring solution obtained bymixing: a)a separated solution obtained by subjecting the bran picklesflavoring solution of any of claims 14-16 to solid-liquid separation, orb)a separated solution obtained by culturing a microorganism having theability to produce lactic acid and a microorganism having the ability toproduce propionic acid in a bran suspension or a treated bransuspension, and then subjecting the obtained culture to solid-liquidseparation; with c) an extract obtained by culturing a microorganismhaving the ability to produce lactic acid and a microorganism having theability to produce propionic acid in a bran suspension or a treated bransuspension, subjecting the obtained culture to solid-liquid separation,and then extracting the obtained residue with an acidic aqueous solutionof ethanol, or d) an extract obtained by culturing a microorganismhaving the ability to produce γ-dodecalactone and/or γ-dodecelactone,subjecting the obtained culture to solid-liquid separation, and thenextracting the obtained residue with an acidic aqueous solution ofethanol.
 22. A clear bran pickles flavoring solution which containspropionic acid in an amount of 500 ppm or more and γ-dodecalactoneand/or γ-dodecelactone in an amount of 20 ppm or more as bran picklesflavoring components, and which has the turbidity of 0.4 or less at 660nm when diluted 10 times with water.
 23. A clear bran pickling solutionwhich contains 50-2000 ppm propionic acid and 1-150 ppm γ-dodecalactoneand/or γ-dodecelactone as bran pickles flavoring components, and whichhas the turbidity of 0.4 or less at 660 nm.
 24. A process for producinga lactone-containing solution, which comprises subjecting a culture of amicroorganism having the ability to produce a lactone to solid-liquidseparation, recovering the residue, and then extracting the lactone fromsaid residue with an acidic aqueous solution of ethanol.
 25. The processaccording to claim 21 or 24, wherein said acidic aqueous solution ofethanol is in the pH range of 1.0-6.5.
 26. The process according toclaim 21, 24 or 25, wherein said acidic aqueous solution of ethanolcontains ethanol in an amount of 10 vol % or more.
 27. A process forproducing pickles, which comprises pickling foods in the bran picklesflavoring solution or the bran pickling solution of any of claims 14-17and 21-23.
 28. Pickles obtained by the process of claim
 27. 29. A seedstrain for rice bran paste comprising a bacterium belonging to the genusCorynebacterium.
 30. A process for producing rice bran paste containingγ-dodecalactone and/or γ-dodecelactone, which comprises adding the seedstrain for rice bran paste of claim 29 to rice bran paste.